Development of antitumor drugs to enhance apoptotic cell death and clinical application

在临床上，实体瘤细胞与抗癌药物和放射的组合治疗被广泛用于增强细胞杀伤。1-（3-C-Ethynyl-β-D-rijbo-pentofuranosyl）cytosine（ECyd）是一种新开发的通过抑制RNA合成诱导细胞死亡的抗癌药物。在这项研究中，我们研究了是否暴露于X射线的人胃腺癌MKN 45细胞在ECyd的存在下，在有限的浓度范围内，没有凋亡诱导细胞凋亡死亡。照射前用0.1μM ECyd处理MKN 45细胞1 h。20戈伊照射后，荧光显微镜结合碘化丙啶染色或电子显微镜观察凋亡和肿胀细胞的形态学区别，并进行评分。当细胞单独用ECyd处理时，仅出现5%的凋亡或坏死细胞。当细胞单独暴露于X射线时，约5%的细胞出现凋亡，约45%的细胞出现肿胀。而在0.1μM ECyd存在下，X射线照射细胞，约25%的细胞为凋亡细胞，约10%的细胞为肿胀细胞。Ac-DEVD-CHO（caspase 3抑制剂）或TPCK（糜蛋白酶样蛋白酶抑制剂）可显著降低ECyd和X射线诱导的细胞凋亡。这些结果表明，caspase 3和糜蛋白酶样蛋白酶是负责共同处理ECyd和X射线诱导的细胞凋亡。在这项研究中，它被证明，共同处理的MKN 45细胞与ECyd和X射线激活G2期相关的凋亡信号与caspase 3和糜蛋白酶样蛋白酶。这些数据可能为开发放射联合抗癌药物治疗实体瘤的临床治疗提供有用的信息。

Clinically, the combination treatment of solid tumor cells with an anticancer drug and radiation was widely used to enhance cell killing. 1-(3-C-Ethynyl-β-D-rijbo-pentofuranosyl) cytosine (ECyd) was newly developed as an anticancer drug to induce cell death by inhibiting RNA synthesis. In this study, we examined whether the exposure of human gastric adenocarcinoma MKN45 cells to X rays in the presence of ECyd at the limited concentration ranges with no apoptosis induced apoptotic cell death. MKN45 cells were treated with 0.1μM ECyd for 1 h before irradiation. After irradiation with 20 Gy, apoptotic as well as swelling cells were morphologically discriminated by fluorescence microscopy combined with propidium iodide staining or electron microscopy and were scored. When cells were treated with ECyd alone, only 5% of either apoptotic or necrotic cells appeared. When cells were exposed to X rays alone, about 5% of apoptotic and about 45% of swelling cells appeared. However, when cells were exposed to X rays in the presence of 0.1μM ECyd, about 25% of totali cells was apoptotic cells and about 10% of total cells was swelling cells. Apoptosis induced by treating cells with ECyd and X irradiation was significantly reduced by the treatment with Ac-DEVD-CHO (caspase 3 inhibitor) or TPCK (chymotrypsin-like protease inhibitor). These results suggested that caspase 3 and chymotrypsin-like proteases were responsible for apoptosis induced by co-treatment with ECyd and X rays. In this study, it was demonstrated that co-treatment of MKN45 cells with ECyd and X rays activated G2-phase-linked apoptotic signaling associated with caspase 3 and chymotrypsin like protease. These data may provide useful information to develop clinical treatment of radiation combined with anticancer drugs for solid tumors.